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Quantitative In Situ Hybridization Histochemistry

  • Andrew Levy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 72)

Abstract

The key advantage of in situ hybridization histochemistry with radiolabeled as opposed to nonradiolabeled probes is that relative differences in the amount of specific mRNA transcripts present in tissue sections can be accurately and reproducibly quantified. Precise quantification and localization of transcripts, as well as the enhanced sensitivity of 35S-labeled probes, allows powerful qualitative as well as quantitative controls to be built into experiments. Interpretation of autoradiographs of tissue sections hybridized in situ depends on careful selection of control tissue and on the availability of a suitable computerized densitometer. Major limiting factors on the performance of in situ hybridization are the relative and particularly the absolute abundance of target transcripts. In situ hybridization on frozen sections produces higher signal intensity and lower background than in situ hybridization on wax-embedded sections, at the expense of tissue morphology (see Fig. 1).
Fig. 1.

Comparison of in situ hybridization on frozen sections and wax-embedded tissue. Macroscopic autoradiographs of 5 µm-thick wax-embedded (A) and 12 µm-thick frozen (B) coronal sections of rainbow trout brain hybridized to a probe complementary to melanin concentrating hormone (MCH2). Sections were treated similarly throughout (apart from the prehybridization protocols) and were exposed simultaneously to adjacent areas of the same sheet of Hyperfilm for 48 h. It can be seen that specific signal is higher and background binding lower (negligible) in the frozen section autoradiograph. The scale marks at the top of frame (B) are millimeter marks cast by the edge of a plastic rule when the image was captured using the program “NIH Image” running on the computer system described in the text.

Keywords

Autoradiography Film Hybridization Histochemistry Autoradiographic Image Apple Macintosh Computer Tube Camera 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copyright information

© Humana Press Inc., Totowa, NJ 1997

Authors and Affiliations

  • Andrew Levy
    • 1
  1. 1.Department of Medicine, Bristol Royal InfirmaryUniversity of BristolUK

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